Rhesus macaques (Macaca mulatta) and Cynomolgus macaques (Macaca fascicularis), are widely used as models of disease pathogenesis in humans and for evaluating vaccines and therapeutics against microbial pathogens, including emerging diseases and potential bioterror pathogens. The ability to interpret the results of these studies is hampered by a limited capacity to accurately quantitate their cellular immune responses. This proposal is focused on developing technology and tools to evaluate cell-mediated immunity in Chinese-origin rhesus macaques and Cynomolgus macaques. Our strategy relies on the identification of common major histocompatibility (MHC) class I alleles, determining their peptide binding motifs, developing bioinformatic tools to predict MHC:peptide interactions, and validating our approach by the identification of Lassa virus, Marburg virus and influenza-derived epitopes. In Specific Aim 1, we will define peptide binding motifs for five common MHC class I molecules each from Cynomolgus macaques and Chinese rhesus macaques. Common A and B loci MHC class I allele frequency data in animal cohorts available for use in experimental research will be provided by collaborators and data generated in-house. A stable transfectant will be produced for each of the five most frequently expressed MHC allele studied, allowing the production and purification of soluble MHC. We will then establish quantitative peptide:MHC peptide binding assays and determine allele specific MHC binding motifs. This quantitative MHC:peptide binding data will be used for algorithm development. In Specific Aim 2, we will develop and make MHC:peptide prediction algorithms available to the scientific community. The large amount of binding data obtained as a result of Specific Aim 1 will be utilized to generate algorithms to predict the MHC binding capacity of peptides. These tools will permit rapid predictions of potential Chinese-origin and Cynomolgus macaque epitopes from any Category A-C pathogen of interest. All binding data and algorithms will be submitted to the Immune Epitope Database, thereby making these tools freely available to the scientific community. In Specific Aim 3, we will experimentally validate our approach in the Lassa, Marburg and influenza virus model systems. We will test the immunogenicity of epitopes derived from Lassa, Marburg and influenza viruses identified in the course of the studies performed in Specific Aim 1 and 2. We now extended our efforts to encompass not only Lassa virus, as proposed in the original submission, but also Marburg and influenza viruses due to improvements in the quality of our computational algorithms. These studies will be performed in collaboration with collaborators. These experiments will validate the approach and tools developed and also identify epitopes that can be utilized to monitor responses in these A-C pathogen systems. Project Narrative: Rhesus macaques and Cynomolgus macaques are widely used as models of disease pathogenesis in humans and for evaluating vaccines and therapeutics against microbial pathogens, including emerging diseases and potential bioterror pathogens. The ability to interpret the results of these studies is hampered by a limited capacity to accurately quantitate their cellular immune responses. This proposal is focused on developing technology and tools to evaluate cell-mediated immunity in these animal models.